To examine more directly the processes o f hair cell loss and epithelial repair, organotypic cultures o f the explants o f the vestibular sensory epithelia from mature mammalian animals were established and developed. The vestibular utricular and saccular maculae from mature guinea pigs and gerbils were dissected out and maintained in organotypic cultures, using procedures essentially the same as those developed by Dr Guy Richardson for neonatal mouse organ o f Corti (Richardson and Russell, 1991).
4.2.1 Preparations
All glassware and instruments were dry heat sterilised one day before use in a OS 150 sterilizer at 180 ° C for two hours. An autoclave 280EH machine or “Little Sister” were used to autoclave all other materials, e.g. rubber, distilled water and vaseline, for 30-40 minutes at 120 ° C at 10 pounds pressure. On the same day before the dissection, the stage o f the laminar flow hood and the dissection microscope were wiped with 80% ethanol. In case o f accidental contamination during dissection, the instruments were dry heat sterilised in a portable hot bead sterilizer (Steri 250, InterFocus Ltd).
H ank’s balanced salt solution (HBSS from Sigma Chemical Ltd), was modified with sodium bicarbonate, without phenol red. Immediately before use, one millilitre o f 1 M HEPES buffer (Sigma Chemical Ltd) was added to 100 ml HBSS to obtain buffered HBSS at pH 7.3. This solution was used to rinse specimens after 80% ethanol sterilisation and used as the dissection medium.
Minimum essential medium (MEM, from Gibco, Life Technologies) with Earle’s salts was used as the basic culture medium throughout the incubation period. M EM was supplemented with heat inactived horse serum (Gibco, Life Technologies). Before mixing M EM with heat inactived horse serum, one millilitre o f 200 mM L-glutamine (Sigma Chemical Ltd) and one millilitre o f 1 M HEPES were added to 100 ml MEM. Then, 10 ml o f heat inactived horse serum was added to 90 ml o f the modified and buffered M EM to provide a culture medium containing 10% heat inactived horse serum.
Rat tail collagen, which was kindly provided by Dr Richardson, was used to coat glass coverslips to ensure attachment o f explanted tissues. To prepare collagen coated coverslips, one and half drops o f collagen solution were placed on a 22 mm diameter round glass coverslip and spread with the tip o f the glass Pasteur pipette. The coated coverslips were exposed to 25% ammonia solution (NH3) for 15 minutes in the fume
cupboard to gel the collagen solution and then rinsed in sterile distilled w ater to remove ammonia and finally stored in a Columbia staining jar containing 8 ml modified H ESS and 3-4 drops o f heat inactived horse serum at room temperature until use.
4.2.2 Tissue Culture Establishment
Pigmented guinea pigs o f either gender and o f ages from five weeks up to 13 months were used to collect inner ear vestibular sensory epithelia. Mature gerbils, eight month old, were also used to collect inner ear vestibular tissues for organotypic cultures to compare the response to gentamicin treatment in different species. The animals were killed by anaesthetic overdose and decapitated. Both bullae were removed from the head under aseptic conditions and the intact bullae were rapidly immersed in cold 80% ethanol
solution to sterilise for 10 minutes. Dissections were performed using a stereo microscope inside a laminar flow cabinet. The specimens were washed twice in cold buffered H ank’s balanced salt solution, and the utricle and saccule from each ear were dissected out on top o f a cold plate to keep specimens cool and reduce autolysis. The dissected tissues were transferred into a small petri dish with fresh HBSS for fine dissection. The membranous sacs were opened and the otoconia layer was removed with a pair o f fine tweezers.
4.2.3 Tissue Culture Maintenance
The vestibular tissues were initially maintained in Maximow slide assemblies. These offer the investigator a means to continuously observe and photographically record active events in the cultured tissues. To set up the Maximow slide assembly, the collagen coated coverslip was centred face up on one 44 mm square coverslip on a piece o f black paper in a large petri dish. With a Pasteur pipette, the prepared tissues were transferred and explanted on to the collagen surface o f the round coverslip with the apical surface o f the epithelium uppermost. The explanted tissues were fed with a single drop (50 ]i\) o f M EM culture medium, then enclosed in the Maximow depression slide which was sealed with a paraffin vaseline mixture. The cultures were maintained in the lying drop position and incubated at 37 “ C incubator (Heraeus Instruments) for 24 or 48 hours. During the whole period in vitro, the cultures were removed from the incubator to observe the development o f the explants, to check for microbial contamination and to photograph under a Nikon inverted light microscope (Diaphot 200) at various times.
4.2.4 Tissue Culture Treatments
After settlement for 24 or 48 hours in vitro, the Maximow slide assembly was split open, and the round coverslip with the explanted cultures was transferred into a 35 mm petri
dish. From this point, some cultured explants were incubated in the agent o f interest. In parallel with in vivo studies, gentamicin sulphate powder (Sigma Chemical Ltd) dissolved in 9:1 MEM culture medium at final concentration 1 mM or 2 mM was directly applied to cultures at 37 “ C to induce hair cell damage in vitro. Twenty four guinea pigs were used to set up vestibular tissue cultures for electron microscopy. To examine gentamicin toxicity, 23 cultures were treated with 1 mM gentamicin for 4, 6 or 24 hours, another 9 cultures were incubated in 2 mM gentamicin for 24 hours and 16 cultures were used as controls. At the end o f the drug treatment, the cultures were rinsed in drug fi'ee culture medium three times, then they were maintained in 250-300 juil o f normal M EM and horse serum culture medium for further periods o f up to 14 days (total 16 days in vitro). Control cultures were fed in normal MEM and horse serum culture medium without gentamicin immediately on transfer to the petri dishes and cultured in the same condition parallel with the treated specimens. The culture medium was changed every two days and the incubator temperature was 37 ° C with constant 5% CO2 supply.
The vestibular utricular cultures from another seven guinea pigs were treated with 1 mM gentamicin for 24 hours, then the cultured utricles were rinsed and continuously grown in gentamicin free medium but containing 3 pg /ml BrdU (Warchol et al., 1993) up to 16 days. Finally, these vestibular utricles incubated with BrdU were processed for BrdU fluorescence labelling.
To test different mammalian species in response to aminoglycoside gentamicin in the culturing system, vestibular tissues fi'om nine mature gerbils were used and maintained in vitro as the procedures for guinea pig cultures. After 24 hours settlement, 12 utricles were
incubated with ImM gentamicin for six hours, then rinsed and cultured in drug free medium. The other six utricles were used as controls. By four days in vitro, six treated and three control cultures were processed for TEM and other cultures survived up to 13 days in culture medium containing 3 pg /ml BrdU in vitro. Finally, these BrdU incubated gerbil cultures were processed for routine and BrdU immunoelectron microscopy.
The vestibular utricles from 32 mature guinea pigs were used to set up cultures for the apoptotic nucleus labelling. After 44 hours settlement in vitro, control cultures were incubated in normal MEM medium with 10% horse serum without gentamicin. The treated cultures were added to the above culture medium containing 2 mM gentamicin sulphate for 6 hours or 24 hours incubation at 37°C, then the gentamicin medium was washed off. Some utricular maculae were processed for apoptotic nucleus labelling by light microscopy immediately after gentamicin treatment. The other cultures were allowed to grow in gentamicin free medium for five more days.